Abstract
The genus Alternaria, functioning as a saprobe, endophyte, and plant pathogen, is widely distributed across various natural and human-impacted environments. Leaf spot and black spot diseases, caused by Alternaria species, are the most prevalent plant diseases within this genus, leading to significant reductions in crop yields and substantial economic losses. To facilitate the timely detection of Alternaria species during the early stages of infection, enable targeted treatments, and mitigate associated damages, we employed a species identification method based on Analysis of whole-GEnome (AGE). We downloaded 148 genomes, including 31 Alternaria species, from the NCBI GenBank database. Through bioinformatics analysis, we constructed a specific-target sequence library and selected a representative sequence per species. The specific target sequences of the seven exemplary Alternaria species were subsequently used for validation and rapid detection, utilizing Sanger sequencing and CRISPR-Cas12a technology, respectively. The results demonstrated that our method accurately identified the target species. Additionally, by combining Enzymatic Recombinase Amplification (ERA) with CRISPR-Cas12a, we achieved rapid and precise identification of genomic DNA samples, with a detection limit as low as 0.01 ng/µL within 30 min. Therefore, AGE proves to be a highly robust and efficient method for the detection of Alternaria species, offering broad potential for various applications.